7kn plasmid

Introduction 

  The 7kb plasmid is being used for an isolation, transformation, and reintroduced into a different ba terrain. The use of this plasmid will be to use a smaller plasmid to introduce gnenes easier into a larger plasmid. 

Methods

  Taking 3ml of culture in 6 different tubes. This pellett was then put through 2 washes of multibuffer and mixed with lysozyme and set to incubate in a heating block. Then this culture in lysozyme was put through the zymo zippy kit isolation. Another kit was used in the second set, the thermoscience plasmid isolation kit. 

Following this protocal:

3ml of culture is pelletier into a 1.5 mL epindorph tube. From here there is then added with 500 microliters of multi buffer and pipette up and down and letting incubate for 5 minutes before centrifuging for 1 minute. The adding of 500 microlitters of multi buffer is added and then pipette up and down, and centrifuge for 1 min. Adding 600 microlitters of lysozyme and pipette up and down then for 15 minutes in a heatblcok at 37*C. Then adding 250 microlitters of resurrection buffer, and 250 microlitters of lysis buffer then inverted for 4-6 times, and 350 microlitters added invert 4-6 times, then centrifuge for 5 minutes, transfer supernatant into filter and centrifuge for 1 minutes, 500 microlitters of wash buffer and centrifuge for 45 seconds, this process was repeated, then 1 minute of additional centrifuge, put filter into another tube and elite in 30-50 microlitters and incubate for 2 minutes followed by 2 minutes of centrifuge.  

This was then taken to the nanodrop and had the purity and the nanograms per microlitters. 

Results

  The first isolation was two samples with a mixture of 9ml of culture with the zymo zippy kit

     ng/ul. 260/280. 260/280

A. 18.5. 1.84. 0.97

B 13.2 1.93 1.1

The second isolation had 2 samples wit a mixture of 6ml of culture and then put through the thermosciencekit. 

 ng/ul 260/280 260/280

A 20 1.69 0.8

B 18.6 1.67 0.81

Discussion 

  The first set of isolations was not a good set of Data through the nanodrop. So a second kit was used and the numbers of the nanodrop were not as bad as the first. This set was sent trough to gel electrophoresis and was found at 1400 kb band with a ladder. So this shows that we have isolated the correct plasmid even if it is still diamerized in the plasmid. This shows that there needs to be some changes going forward we will add a lysozyme of 30 minutes instead of 15 and if this does not work then a different kit will be used.